Phytochemicals for treatment of endometriosis

ABSTRACT

New compositions are disclosed that comprise the phytochemical Diindolylmethane (DIM), as well as its precursor, Indole-3-carbinol (I3C), and cogener, 2-(Indol-3-ylmethyl)-3,3′diindolylmethane (LTR-1), acceptable carriers and/or excipients. These compositions are administered to prevent or reduce symptoms associated with mastalgia and endometriosis.

This application is a continuation of, and claims benefit of, U.S.application Ser. No. 09/404,111, filed Sep. 23, 1999, now U.S. Pat. No.6,689,387 and is incorporated by reference in its entirety herein.

1. INTRODUCTION

The present invention relates to compositions and methods for preventionor reduction of symptoms associated with mastalgia and endometriosis byadministering phytochemicals. Among the phytochemicals useful in thecompositions and methods of the invention are dietary indole,Diindolylmethane (DIM), as well as its precursor, Indole-3-carbinol(I3C), and cogener, 2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR-1).

2. BACKGROUND OF THE INVENTION

2.1 Mastalgia

In the typical life-cyle of modern women, the mid-thirties until thecessation of menstrual periods is defined as “peri-menopause”. Women areclassified as perimenopausal if menses have been experienced in the last12 months, but with irregularity or changes in menstrual flow. Duringthis stage of life women suffer increasing incidence of both recurrentbreast pain, or “mastalgia”, and endometriosis, describing the painfulcondition of persistence of abnormal endometrial tissue in the abdominalcavity. These two conditions, common to the perimenopause, are poorlyunderstood and presently lack medical therapy that is both effective andreasonably free of side effects (Prior, J. C., “Perimenopause: thecomplex endocrinology of the menopausal transition”, Endocr. Rev., 19,pages 397-428, 1998).

While a contributing role of estrogen status has been suspected in theseconditions, few consistent abnormalities in endocrine status have beenidentified. Circulating estrogen levels are normal in both mastalgia andendometriosis patients. There is accumulating evidence that describesdiminished progesterone production during the perimenopause that maycreate a relative “dominance” in the activity of estrogen. However, noone theory or endocrine imbalance explains the occurrence of mastalgiaand endometriosis. (Santoro, N., Rosenberg, J., et al.,“Characteristicsof reproductive hormonal dynamics in the perimenopause”, J. ClinicalEndocrinology and Metab., 81, pages 1495-501, 1996).

Recurrent, cyclical breast pain or “mastalgia” is one of the most commonreasons for women's visits to their doctors. It has been estimated that50-70% of all women experience significant mastalgia at some point intheir life. In its most common form, mastalgia is a chronic condition ofrecurring pain, which is worse during the few days before menses (Blue,J., Harman, J., et al., “Mastalgia review: St. Marks Breast Centre”, NewZealand Medical Journal, 111(1059), pages 33-34, 1998). Traditionally,treatment choices for mastalgia have ranged from dietary manipulation(caffeine, fat, and alcohol reduction) or evening primrose oil tohormonal medications (bromocriptine and danazol) for severe breast pain.Bromocriptine (Parlodel) and danazol have a response rate of 70%, buthave reported adverse side effects of up to 30-35% (Gateley, C. A. andMansen, R. E., “Management of the painful and nodular breast”, BritishMedical Bulletin, 47, 284-94, 1991; Nazli K., et al. Controlled trial ofthe prolactin inhibitor bromocriptine (Parlodel) in the treatment ofsevere cyclical mastalgia. British Journal of Clinical Practice. 1989;43(9): 322-7; Kontosolis K. et al., Comparison of tamoxifen with danazolfor treatment of cyclical mastalgia. Gynecol. Endolcrinol. 1997; 11,page 393-397). The use of medroxyprogesterone acetate to support levelsof progesterone, possibly low in this condition, proved relief no betterthan placebo in a controlled trial. (Maddox, P. R., Harrison, B. J., etal., “A randomized controlled trial of medroxyprogesterone acetate inmastalgia”, Annals of the Royal College of surgeons of England, 72(2),pages 71-6, 1990).

The approach of dietary supplementation for mastalgia has been exploredby earlier investigators. This included the addition of high doses ofevening primrose oil, beta carotene, and vitamin A to the diet ofaffected women. Evening primrose oil is used by British physicians as aninitial intervention to control mastalgia because of its non-hormonalcomposition. Though it has been found to normalize the ratio ofessential fatty acids to saturated fatty acids in the serum of womenwith mastalgia, the therapy requires 3 to 4 months for benefit.Improvements were seen in up to 40% of patients but side effectsincluded bloating and nausea (Maddox, P. R., “The management ofmastalgia in the UK”, Hormone Research, 32, pages 21-27, 1989). Italianresearchers explored the addition of combinations of beta carotene andVitamin A (retinol) in the management of mastalgia. (Santamaria L,Dell'Orti, M., et al., “Beta-carotene supplementation associated withintermittent retinol administration in the treatment of pre-menopausalmastodynia,”, Boll Chim Far, 128, pages 284-287, 1989). Some success wasreported, but the high doses of retinol required (150-300,000 I.U perday) are in the range associated with significant side effects whichinclude headache, skin lip and mouth dryness, nausea, dizziness, andalopecia. Based on the common occurrence of mastalgia as a disorder inwomen, the need exists for more effective therapy with acceptable risksand side effects (Ashley B., “Mastalgia”, Lippincotts Primary CarePractice. 1998; 2(2): 189-93).

2.2 Endometriosis

Endometriosis is a disease that affects as many as 15% of fertile womenand up to 50% of infertile women. Its occurrence increases with age andis greatest in the perimenopausal years (Tzingounis V A, and CardamakisE., “Modern approach to endometriosis”, Annals of the New York Academyof Sciences, 816, pages 320-330, 1997). Endometriosis refers to thepresence of functional endometrial glands and stroma in abnormallocations outside the uterine cavity. Despite extensive research, thenatural history and pathogenisis of endometriosis is still poorlyunderstood and remains controversial. As with mastalgia, mosttherapeutic approaches have been directed at hormonal therapy. The mostcommon therapy involves the use of danazol. Danazol is a syntheticsteroid with androgenic action suppressing the pituitary gland cyclingnecessary for menstrual periods. Amenorrhea, or lack of menstrualperiods results. Though providing some relief from the pain ofendometriosis adverse side effects are experienced in up to 80% of womenusing Danazol (Greenblatt R B, Dmowski, W. P., et al. “Clinical studieswith an anti-gonadotropin—danazol”, Fertil Steril, 22, page 102, 1971).Notably these side effects include weight gain, fluid retention, acne,decreased breast size, hot flushes and mood changes. In addition todanazol, other hormonal manipulations used in the management ofendometriosis involve use of gonadotropin releasing hormone analogues(GnRH) and the drug gestrinone, a synthetic steroid derived from19-nortestosterone. The side effects, associated with these therapiesare significant and include the spectrum of symptoms associated withhypoestrogenism and menopause. These include hot flushes, night sweats,and osteoporosis. (Telimaa, E. J., Puolakka, J., et al.,“Placebo-controlled comparison of danazol and high-dosemedroxyprogesterone acetate in the treatment of endometriosis”, GynecolEndocrinol, 1, page 51, 1987, and Thomas E. J., Cooke, I. D., et al.,“Impact of gestrinone on the course of asymptomatic endometriosis”, Br.Med J., 294, page 272, 1987). Clearly, more benign approaches to themanagement of the pain of endometriosis are needed.

2.3 Dietary Indoles

Diindolylmethane (DIM), as well as its precursor, Indole-3-carbinol(I3C), and cogener, 2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR-1)are natural phytochemicals and part of the family of dietary indolesdiscovered in cruciferous vegetables. DIM and I3C are found incruciferous vegetables including broccoli, cauliflower, cabbage andBrussels sprouts (Bradfield C A and Bjeldanes L F, High performanceliquid chromatographic analysis of anticarcinogenic indoles in Brassicaoleracea. J Agric. Food Chem. 1987; 35:46-49). DIM, together with thelinear trimer, LTR-1, are formed from the precursor indole, I3C, afterthe enzymatic release of I3C from parent glucosinolates found in allcruciferous vegetables.

It is now known that there is a connection between dietary cruciferousindoles and estrogen metabolism. H. Leon Bradlow, Ph.D. and his group atthe Strang Cancer Prevention Laboratory in New York were the first toestablish the link between phytonutrients from cruciferous vegetablesand estrogen metabolism. They showed that supplemental use of a I3C canact to promote a dramatic change in the metabolism of estrogenMichnovicz J J, et al., Changes in levels of urinary estrogenmetabolites after oral indole-3-carbinol treatment in humans. J NatlCancer Inst. 1997 May 21; 89(10):718-23. This change in metabolism hasthe power to greatly reduce estrogen exposure as a risk for cancer andprovides a dietary approach to improving estrogen metabolism. Whencruciferous phytochemicals are added to the diet its metabolism isshifted. This produces a predominance of 2-hydroxy and2-methoxyestrogens (Michnovicz J J, et. al., Changes in levels ofurinary estrogen metabolites after oral indole-3-carbinol treatment inhumans. J Natl Cancer Inst. 1997 May 21; 89(10):718-23). An increasedproportion of 2-hydroxy metabolites is correlated to protection frombreast cancer. This relationship has been documented in case-controlstudies (Ho G H, et al. Urinary 2/16 alpha-hydroxyestrone ratio:correlation with serum insulin-like growth factor binding protein-3 anda potential biomarker of breast cancer risk. Ann Acad Med Singapore1998; 27:294-299, and Schneider J., et al., Abnormal oxidativemetabolism of estradiol in women with breast cancer. Proc Natl Acad SciUSA 1982; 79: 3047-3051). The 2-hydroxy metabolites have been called“good estrogens (Bradlow H L, et al., 2-hydroxyestrone: the ‘good’estrogen. J Endocrinol. 1996 September; 150 Suppl:S259-65), and mayfunction as antioxidants (Komura S, et al., Catecholestrogen as anatural antioxidant. Ann N Y Acad Sci. 1996 Jun 15; 786:419-429).

With regard to prior art and the dietary indoles, the supplemental useof I3C, which converts to DIM and LTR-1 after passage through thestomach, has been the subject of a U.S. Pat. No. 5,895,787) describingthe use of I3C and related dietary indoles to reduce the symptoms offibromyalgia. Despite this use, no relationship between fibromyalgia andestrogen status has been documented (Bengtsson A., Henriksson, K. G.,“Primary fibromyalgia. A clinical and laboratory study of 55 patients.”,Scand J. Rheumatol, 15(3) pages 340-7, 1986) Published reports havedemonstrated the usefulness of dietary supplementation with I3C inrecurrent laryngeal papillomatosis and cervical dysplasia. (Rosen, C.A., Woodson, G. E. et al., “Preliminary results of the use ofindole-3-carbinol for recurrent respiratory papillomatosis,Otolaryngology Head Neck Surgery, 118, pages 810-5, 1998, and Jin L.,Qi, M., et al., “Indole-3-carbinol prevents cervical cancer in humanpapilloma virus type 16 (HPV16) transgenic mice”, Cancer Research,59(16), pages 3991-7, 1999) These are both diseases related to theaction of the human papilloma virus and may represent conditionssensitive to estrogen metabolic status. Use of DIM and LTR-1 inabsorption enhancing formulations for improving the balance of estrogenmetabolites has been the subject of earlier investigation by the presentinventor and provides the basis of U.S. patent application Ser. No.09/053,180. This prior work has allowed for the present investigation ofthe use dietary indoles as dietary supplements to beneficially impactmastalgia and endometriosis.

While previous work with dietary indoles, like DIM, has indicated theirability to impede the growth of breast cancer in animals (Chen, I.,McDougal, A., et al., “Aryl hydrocarbon receptor-mediated antiestrogenicand antitumorigenic activity of diindolylmethane,” Carcinogenesis,19(9), 1631-9, 1998) no reports exist as to the usefulness ofcruciferous phytochemicals in managing mastalgia or endometriosis.

3. SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for preventionor reduction of symptoms associated with mastalgia and endometriosis byadministering phytochemicals, e.g., dietary indoles. In a preferredembodiment, the pain associated with endometriosis and mastalgia isprevented or reduced. In another embodiment, the presence of a markerassociated with endometriosis is reduced through phytochemicaltreatment. Among the phytochemicals useful in the compositions andmethods of the invention are dietary indole, Diindolylmethane (DIM), aswell as its precursor, Indole-3-carbinol (I3C), and cogener,2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR-1).

Also according to the present invention, a pharmaceutical composition isprovided, which comprises a phytochemical, preferably I3C, DIM and/orLTR-1, and, optionally, pharmaceutically acceptable carriers.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the observation that administrationof phytochemicals, in particular, I3C, DIM, and LT-1, has improvedsymptoms of mastalgia and endometriosis.

The facilitated delivery of DIM and related indoles as dietarysupplements may be accomplished according to formulations and methodsdescribed in U.S. patent application Ser. No. 09/053,180. Theeffectiveness of supplemental DIM is further supported byco-administration of phytochemicals (e.g., DIM, LTR-1) with grapefruitconcentrate, which additionally facilitates the absorption ofphytochemicals (e.g., DIM, LTR-1).

4.1. Methods of Treating Mastalgia

The invention provides compositions and methods for prevention orreduction of symptoms associated with mastalgia. In a preferredembodiment, breast pain of subjects suffering from mastalgia isprevented, reduced and/or eliminated through the administration ofphytochemicals, e.g., dietary indoles, in a pharmaceutically acceptablefashion. In preferred embodiments, the phytochemicals are DIM, I3C andLTR-1. In particular embodiments, I3C, DIM, or LTR-1, alone or incombination with each other or other dietary supplements, areadministered orally in, for example, the form of encapsulated dietarysupplements.

The I3C is preferably administered at a dose of 200-500 mg per day. Inalternative embodiments, I3C is administered at doses of 200-300 mg perday, 300-400 mg per day and 400-500 mg per day.

DIM is administered providing 150-500 mg per day of DIM. In preferredembodiments, the dose of DIM, I3C or LTR-1 is 150-200 mg per day,200-300 mg per day, 300-400 mg per day, and 400-500 mg per day.

In a preferred embodiment, DIM is administered in an absorptionenhancing formulation, as described in U.S. patent application Ser. No.09/053,180, providing 60-500 mg per day of DIM suspended asmicroparticles in a starch carrier matrix. In preferred embodiments, thedose of processed DIM is 60-100 mg per day, 100-200 mg per day, 200-300mg per day, 300-400 mg per day, and 400-500 mg per day.

The LTR-1 is preferably administered in an absorption enhancingformulation providing 60-500 mg per day of LTR-1 suspended asmicroparticles in a starch carrier matrix as previously described,however, the present invention contemplates the administration of anypreparation of LTR-1. In a preferred embodiment, the dose of LTR-1 is150-200 mg per day. In preferred embodiments, the dose of processedLTR-1 is 60-100 mg per day, 200-300 mg per day, 300-400 mg per day, and400-500 mg per day.

Doses of the phytochemicals of the invention can also be calculatedbased upon the body weight of the subject to be treated. Doses ofphytochemicals between 0.5 and 3 mg per kg of body weight per day arepreferred. In another preferred embodiment, the phytochemicals areadministered at a dose of between 0.5 and 2.0 mg per kg per day,preferably 1.5 mg per kg per day.

Alternatively, the co-administration of grapefruit, grapefruitconcentrate, grapefruit juice, or grapefruit juice concentrate, or othergrapefruit-derived composition along with a dietary indole (e.g., I3C,DIM or LTR-1) can be used to increase absorption of the phytochemicalsand promote even more efficient relief from the symptoms of mastalgia.

In an alternative embodiment, the dietary indole (e.g., DIM or LTR-1) isadministered in the form of a liposomal sublingual spray applieddirectly to the oral mucosa. This liposomal suspension providesphytochemical loaded liposomes to administer the phytochemicals andcreate a sustained delivery system. Dietary indole (e.g., DIM or LTR-1)containing liposomes are sequestered in the oral mucosa, allowingabsorption which avoids “first pass” hepatic metabolism. The liposomalspray uses standard liposomal preparation and structural lipidingredients (Ranade, V. V., “Drug delivery systems. 1. Site-specificdrug delivery using liposomes as carriers,” J. Clin. Pharmacol.29(8):685-94, 1989). In a preferred embodiment, the liposomal spray isadministered at a dose of 5-30 mg of dietary indole (e.g., DIM or LTR-1)daily delivered in 2-12 sprays of a typical liposomal preparation.

Alternatively the phytochemicals (e.g., DIM or LTR-1) may beadministered in the form of a transdermal cream applied directly to theskin. This cream utilizes various absorption enhancing emollients andconsists of phytochemical (e.g., DIM or LTR-1) in a concentration of1-3% by weight. It is designed as a moisturizing cosmetic that isformulated to allow application directly to painful breasts in women notwishing to take phytochemicals orally. Formulations are also made withthe neurohormone, melatonin, to provide a nighttime cosmetic mosturizeroffering the benefits of melatonin in combination with the phytochemical(e.g., DIM or LTR-1). This allows application of cruciferous indoles andmelatonin directly to underlying breast tissue with the added benefit ofsleep regulating action from melatonin. In a particular embodiment,application of from 5-10 cc of the transdermal preparation daily is usedto administer from 5-30 mg of DIM or other dietary indole per day, andoptionally, 3-10 mg of melatonin per day.

Alternatively, the phytochemical (e.g., DIM or LTR-1) may beadministered in the form of a vaginal cream or suppository containingmicrocrystalline phytochemical (e.g., DIM or LTR-1) in a combined doseof 20-100 mg. This allows application of cruciferous indoles directly tovaginal and cervical mucosa for the benefit of cervical dysplasia.

The phytochemicals of the invention may be administered in anyappropriate amount in any suitable galenic formulation and following anyregime of administration.

The actual administered amount of phytochemical may be decided by asupervising physician and may depend on multiple factors, such as, theage, condition, file history, etc., of the patient in question.

The subject, or patient, to be treated using the methods of theinvention is an animal, e.g., a mammal, and is preferably human, and canbe a fetus, child, or adult. In a preferred embodiment, the subject is ahuman female.

4.2. Methods of Treating Endometriosis

The invention provides compositions and methods for reduction orprevention of symptoms associated with endometriosis. In a preferredembodiment, the pain of subjects suffering from endometriosis isprevented, reduced and/or eliminated through the administration ofphytochemicals in a pharmaceutically acceptable fashion. In anotherpreferred embodiment, the levels of an endometriosis marker (e.g.,Ca-125 antigen, a serum marker of endometriosis) in subjects sufferingfrom endometriosis is lowered through the administration ofphytochemicals in a pharmaceutically acceptable fashion. In preferredembodiments, the phytochemicals are DIM, I3C and LTR-1. In particularembodiments, I3C, DIM, or LTR-1, alone or in combination with each otheror other dietary supplements, are administered orally in, for example,the form of encapsulated dietary supplements.

The 13C is preferably administered at a dose of 200-500 mg per day. Inalternative embodiments, I3C is administered at doses of 200-300 mg perday, 300-400 mg per day and 400-500 mg per day.

DIM is administered providing 30-500 mg per day of DIM. In preferredembodiments, the dose of DIM, I3C or LTR-1 is 30-100 mg per day, 100-200mg per day, 200-300 mg per day, 300-400 mg per day, and 400-500 mg perday.

In a preferred embodiment, DIM is administered in an absorptionenhancing formulation, as described in U.S. patent application Ser. No.09/053,180, providing 30-500 mg per day of DIM suspended asmicroparticles in a starch carrier matrix. In preferred embodiments, thedose of processed DIM is 30-100 mg per day, 100-200 mg per day, 200-300mg per day, 300-400 mg per day, and 400-500 mg per day.

The LTR-1 is preferably administered in an absorption enhancingformulation providing 30-400 mg per day of LTR-1 suspended asmicroparticles in a starch carrier matrix as previously described,however, the present invention contemplates the administration of anypreparation of LTR-1. In a preferred embodiment, the dose of LTR-1 is100-200 mg per day. In preferred embodiments, the dose of processedLTR-1 is 30-100 mg per day, 200-300 mg per day, 300-400 mg per day, and400-500 mg per day.

Doses of the phytochemicals of the invention can also be calculatedbased upon the body weight of the subject to be treated. Doses ofphytochemicals between 1 and 3 mg per kg of body weight per day arepreferred. In another preferred embodiment, the phytochemicals areadministered at a dose of between 1.5 and 2.5 mg per kg per day,preferably 2.0 mg per kg per day.

Alternatively, the co-administration of grapefruit, grapefruitconcentrate, grapefruit juice, or grapefruit juice concentrate, or othergrapefruit-derived composition along with I3C, DIM or LTR-1 can be usedto increase absorption of the phytochemicals and promote even moreefficient relief from the symptoms of endometriosis, including thereduction of markers associated with endometriosis.

In an alternative embodiment, the phytochemical (e.g., DIM or LTR-1) isadministered in the form of a liposomal sublingual spray applieddirectly to the oral mucosa. This liposomal suspension providesphytochemical loaded liposomes to administer the phytochemicals andcreate a sustained delivery system. DIM and LTR-1 containing liposomesare sequestered in the oral mucosa, allowing absorption which avoids“first pass” hepatic metabolism. The liposomal spray uses standardliposomal preparation and structural lipid ingredients. (Ranade, V. V.,“Drug delivery systems. 1. Site-specific drug delivery using liposomesas carriers, J. Clin. Pharmacol. 29(8):685-94, 1989; Crommelin, D. J. A.and Schreir, H., “Liposomes”, Colloidal Drug Delivery Systems, Kreuter,J. editor, Marcel Dekker, N.Y., 1994, p. 85). In a preferred embodiment,the liposomal spray is administered at a dose of 5-30 mg ofphytochemical daily delivered in 2-12 sprays of a typical liposomalpreparation.

Alternatively the phytochemicals (e.g., DIM or LTR-1) may beadministered in the form of a transdermal cream applied directly to theskin. This cream utilizes various absorption-enhancing emollients andconsists of DIM or LTR-1 in a concentration of 1-3% by weight. It isdesigned as a moisturizing cosmetic which is formulated to allowapplication directly to the skin of women not wishing to takephytochemicals orally. Formulations are also made with the neurohormone,melatonin, to provide a nighttime cosmetic moisturizer offering thebenefits of melatonin in combination with the phytochemical (e.g., DIMor LTR-1). This provides, the added benefit of sleep regulating actionfrom melatonin. In a particular embodiment, application of from 5-10 ccof the transdermal preparation daily is used to administer from 5-30 mgof phytochemical (e.g., DIM or LTR-1) per day, and optionally, 3-10 mgof melatonin per day.

Alternatively the phytochemical (e.g., DIM or LTR-1) may be administeredin the form of a vaginal cream or suppository containingmicrocrystalline DIM or LTR-1 in a combined dose of 20-100 mg. Thisallows application of cruciferous indoles directly to vaginal andcervical mucosa for the benefit of cervical dysplasia.

The phytochemicals of the invention may be administered in anyappropriate amount in any suitable galenic formulation and following anyregime of administration.

The actual administered amount of phytochemical may be decided by asupervising physician and may depend on multiple factors, such as, theage, condition, file history, etc., of the patient in question.

The subject, or patient, to be treated using the methods of theinvention is an animal, e.g., a mammal, and is preferably human, and canbe a fetus, child, or adult. In a preferred embodiment, the subject is ahuman female.

4.3. Pharmaceutical Compositions

The pharmaceutical compositions according to the present inventionpreferably comprise one or more pharmaceutically acceptable carriers andthe active constituents. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the compositionand not deleterious to the recipient thereof.

It will be appreciated that the amounts of phytochemical required forsaid treatment will vary according to the route of administration, thedisorder to be treated, the condition, age, and file history of thesubject, the galenic formulation of the pharmaceutical composition, etc.

Preferably, the phytochemical used in the invention has been processedto enhance bioavailability, as is described in U.S. patent applicationSer. No. 09/053,180. So processed DIM or LTR-1 is referred to as“processed DIM” and “processed LTR-1”, respectively. However, anysuitable preparation of phytochemical can be used in the methods andcompositions of the invention.

The following is a list of ingredients useful for formulating processedDIM or LTR-1:

-   -   1. About 10 to about 40 percent by weight of LTR-1 or DIM.    -   2. About 10 to about 40 percent by weight of the following,        alone or in combination: vitamin E succinate polyethylene glycol        1000; vitamin E succinate Polyethylene glycols with polyethylene        glycol. (with a molecular weight range of 400-2000); other        polyethylene glycol esters such as those formed by fatty acids        such as oleic acid or stearic acid; polyvinylpyrrolidones;        polyvinylpolypyrrolidones; Poloxamer 188, Tweens; or Spans.    -   3. About 5 to about 20 percent by weight of the following, alone        or in combination: phosphatidyl choline (derived from soy        lecithin and supplied as Phospholipon 50 G from Rhone Poulenc        Rorer); dioleoyl phosphatidylcholine; phoshatidylglycerol;        dioleoylphosphatidylglycerol; dimyristoylphosphatidylcholine;        dipalmitoylphosphatidylcholine; phosphatidylethalolamines;        phosphatidylserines; or sphingomyelins; or other sources of        phospholipids as those from purified Milk Fat Globule Membrane;        glycerolesters; poly glycerol esters; or ethoxylated castor oil.    -   4. About 15 to about 30 percent by weight of the following,        alone or in combination: hexanol; ethanol; butanol; heptanol;        2-methyl-1-pentanol; various ketone solvents that would be        acceptable in foods such as methyl ethyl ketone, acetone and        others; propylene glycol; and certain ester solvents such as        ethyl acetate.    -   5. About 20 to about 40 percent by weight of the following,        alone or in combination: modified starch such as Capsul™ Starch        from National Starch, Inc.; methylcellulose; hydroxypropyl        methylcellulose; hydroxyethylcellulose;        hydroxypropylethylcellulose; pectin; gum arabic; gelatin; or        other polymeric matrix-forming preparation known to those        skilled in the art, soluble in water and, suitable for spray        drying.    -   6. About 0.5 to about 35 percent by weight of the following,        alone or in combination: aerosil 200; or any other flow        enhancing excipient from silica, or related salt, known to those        skilled in the art.

The following is a detailed method of formulating processed DIM:

-   -   1. 6.75 kg of TPGS is heated just beyond its melting point with        constant stirring in a heated vessel (“First vessel”).    -   2. 9.38 kg of hexanol and 9.83 kg of jet milled DIM is added to        the first vessel and the mixture stirred to a uniform suspension        at 70° C. 1.4 kg of phosphatidyl choline is then added.    -   3. In a second larger vessel, 185 L of water and 10.7 kg of        starch are thoroughly mixed with a Cowles blade. This mixture is        neutralized to pH 7 with a small amount of sodium carbonate and        then heated to 75° C. and stirred for 1 hour.    -   4. The contents of the first vessel is added to the starch        mixture in the second larger vessel and thoroughly mixed with a        homogenizing rotor/stator type mixer at moderate speed for 15        minutes.    -   5. The mixture from step 4 is spray dried with a small amount        (approximately 0.5%) of hydrophilic silica to provide a free        flowing powder of finely dispersed microparticles containing the        co-precipitated TPGS, phosphatidyl choline and DIM in an        amorphous, non-crystalline structure.    -   6. The flowable powder product is collected and stored in        evacuated foil sacks, after de-aerating and flushing with        nitrogen.    -   7. Analysis of presence of unchanged dietary ingredient, reveals        about 30 to about 33 percent by weight of DIM.

The procedure of making processed DIM may be summarized as follows:

-   -   (a) heating one or more solubilizing emulsifiers selected from        the group consisting of vitamin E succinate polyethylene glycol        1000, polyvinylpyrrolidone, polyoxyethylene stearate, sodium        cholate, deoxycholate and taurocholate;    -   (b) adding to the product of step (a) a solvent and a surfactant        phospholipid selected from the group consisting of phosphatidyl        choline, dioleoyl phosphatidyl choline, phosphatidylglycerol,        dioleoylphosphatidylglycerol, dimyristoylphosphatidylcholine,        dipalitoylphosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine and sphingomyelin to produce a solution;    -   (c) dissolving in the solution of step (b) LTR-1 and/or DIM;    -   (d) adding to the solution of step (c) a solution containing an        encapsulator;    -   (e) mixing the solution produced in step (d) to produce a        microdispersion with a particle size of 5 microns or less; and    -   (f) spray drying the resulting mixture to leave a solid        hydrophobic phytochemical composition.

In general, a suitable (therapeutically effective) amount of I3C is300-500 mg per day. DIM is preferably administered in an absorptionenhancing formulation, as described in U.S. patent application Ser. No.09/053,180, at 50-200 mg per day as a suspension of microparticles in astarch carrier matrix. The LTR-1 is preferably administered in anabsorption enhancing formulation at 50-200 mg per day as a suspension ofmicroparticles in a starch carrier matrix. The actually administeredamounts of phytochemical may be decided by a supervising physician. Thephytochemicals of the invention may be administered alone or incombination with one another, and/or with other dietary supplements. Thecombinations of phytochemicals and supplements can be in the samecomposition for administering in combination concurrently, or indifferent compositions for administering concurrently but separately, orsequentially.

Therapeutic formulations include those suitable for parenteral(including intramuscular and intravenous), oral, rectal or intradermaladministration, although oral administration is the preferred route.Thus, the pharmaceutical composition may be formulated as tablets,pills, syrups, capsules, suppositories, formulations for transdermalapplication, powders, especially lyophilized powders for reconstitutionwith a carrier for intravenous administration, etc.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; asweetening agent, such as sucrose or saccharin; and/or a flavoringagent, such as peppermint, methyl salicylate, or orange flavoring.

Therapeutic formulations suitable for oral administration, e.g., tabletsand pills, may be obtained by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bymixing phytochemicals, and compressing this mixture in a suitableapparatus into tablets having a suitable size. Prior to the mixing, thephytochemical may be mixed with a binder, a lubricant, an inert diluentand/or a disintegrating agent.

In a preferred embodiment, phytochemical is mixed with a binder, such asmicrocrystalline cellulose, and a surfactant, such as sodium laurylsulphate until a homogeneous mixture is obtained. Subsequently, anotherbinder, such as polyvidone, is transferred to the mixture under stirringwith a small amount of added water. This mixture is passed throughgranulating sieves and dried by desiccation before compression intotablets in a standard tableting apparatus.

A tablet may be coated or uncoated. An uncoated tablet may be scored. Acoated tablet may be coated with sugar, shellac, film or other entericcoating agents.

Therapeutic formulations suitable for parenteral administration includesterile solutions or suspensions of the active constituents. An aqueousor oily carrier may be used. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Formulations for parenteral administrationalso include a lyophilized powder comprising phytochemical that is to bereconstituted by dissolving in a pharmaceutically acceptable carrierthat dissolves said phytochemical.

When the pharmaceutical composition is a capsule, it may contain aliquid carrier, such as a fatty oil, e.g., cacao butter.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. These compositions cantake the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations and the like. Thecomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides.

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In one embodiment of the pharmaceutical composition according to theinvention, two or more active constituents are comprised as separateentities. The two entities may be administered simultaneously orsequentially.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The invention is further explained by the following illustrativeexamples.

5. EXAMPLE Manufacture of Processed Dim or LTR-1 for Enhanced OralBioavailability

Preparation of processed DIM and LTR-1 was accomplished according to thesteps outlined in U.S. patent application Ser. No. 09/053,180. Briefly,this included mixture of about 10-40% by final weight of either DIM orLTR-1 with about 10-40% by final weight of vitamin E polyethylene glycol1000 succinate(Vitamin-E-TPGS, Eastman Chemical), 2-20% by final weight,phosphatidyl choline (Phospholipon 50G, Rhone Poulenc) and 15-30% byfinal weight hexanol. This mixture was made homogeneous by mixing. Thehomogeneous mixture of indoles and other oil soluble substituents listedabove was added to a solution of modified starch in water (Capsul Starchfrom National Starch, Inc.). The starch component forms form from 30-70%of the final dry weight of the product. The well dispersed finalcombined mixture was then subjected to spray drying. The resultantproduct was a fine powder containing either DIM or LTR-1 containedwithin the starch particles.

6. EXAMPLE Manufacture of Capsules Containing 13C, DIM and LTR-1

Pure Indole-3-carbinol (I3C) was obtained from standard suppliers(Sabinsa, Inc or Designed Nutritional Products, Inc.) Capsules Inc.,).Capsules containing 300-500 mg were manufactured by placing that amountof I3C into opaque gelatin capsules.

Capsules containing 150-300 mg of processed DIM, as produced accordingto the steps described in example 1., were made by mixing the processedDIM with microcrystaline cellulose and placing the mixed powder intoopaque gelatin capsules.

Similarly, capsules containing 150-300 mg of processed LTR-1, asproduced according to the steps described in example 1., were made bymixing the processed LTR-1 with microcrystaline cellulose and placingthe mixed powder into opaque gelatin capsules.

7. EXAMPLE Manufacture of DIM or LTR-1 in a Cream for TransdermalDelivery

For the aqueous phase of the emulsion, a mixture of 70 grams ofpropylene glycol and 633 grams of water is heated to 95° C. The oilphase of the emulsion is prepared by heating a mixture of the followingto 105° C.: 30 grams cetostearyl alcohol (Alfol 16/18, Vista), 30 gramshydrogenated soy monoglyceride (Myverol 18-06, Quest), 30 g of a mixtureof polyoxyethylene stearic acid ester and mono- and di-glycerides offatty acids (Arlacel 165, ICI), 10 grams polyethylene (Epolene N-34,Eastman), and 50 g. of squalene. The active ingredient phase is preparedseparately also by gently heating to about 63° C. a mixture of thefollowing to uniformity: 30 g d-α-tocopherol polyethylene glycol 1000succinate (Vitamin E TPGS, Eastman), 50 g isopropyl myristate, and 15 gof DIM or 15 g of LTR-1. The above oil phase is added to the aqueousphase using a rotor/stator type homogenizer at moderate speed. Themixture is cooled to 75° C. and 50 grams of lemon oil is added with lowspeed mixing followed by addition of the active ingredient phase.Lastly, 2 g of a 3:1 mixture of methyl paraben to propyl paraben isadded to the emulsion. This mixture is transferred to the reservoir of ahigh pressure homogenizer such as the Microfluidics& Model 110Y. Theemulsion is passed through the homogenizer approximately five times at15,000 psi operating pressure that is sufficient to form a cream of thedesired consistency which will not separate on standing.

8. EXAMPLE Manufacture of DIM or LTR-1 in a Liposomal Spray forSublingual and Mucosal Delivery

A standard liposomal preparation technique was used to prepare aliposomal suspension of DIM and separately, a liposomal preparation ofLTR-1. Briefly, propylene glycol (7.0 gms) was heated to 92° C. on awater bath, 8 grams of partially hydrogenated pure egg yolk lecithin,and 320 mg of stearylamine were added and dissolved to give a clearliquid. To this liquid was added 500 mg of jet milled DIM. Thistranslucent solution was added to 200 ml of a 1% aqueous solution ofdextran T 40 pre-warmed to 55° C. and the mixture was stirred in apropeller mixer at 50° C. for 3 minutes after which it was cooled toroom temperature. This procedure yielded an off-white, dextran T40/liposome suspension, thus encapsulating the DIM.

Equivalent steps were undertaken to prepare a lipsomal suspensionencapsulating LTR-1.

9. EXAMPLE Use of Processed DIM for Treatment of Mastalgia

The purpose of this study was to determine whether dietarysupplementation with the cruicferous phytochemical, Diindolylmethane(DIM) is effective in relieving the pain of this chronic condition.

B. R. is a 44 year old woman referred for evaluation of perimenopausalsymptoms of chronic recurring breast pain. She had been troubled bybreast pain for many years with recent worsening in her symptoms.

At age 35 she had undergone a vaginal hysterectomy due to excessive andpainful menstrual periods. Since her ovaries were left intact, she hasreceived no Hormonal Replacement Therapy (HRT). During the last 4 yearsshe experienced a monthly cycle of breast pain for about one week ofeach month. The breast pain was bilateral, associated with significanttenderness to touch, and a discomfort described as “heaviness orswelling.” A trial of a women's health supplement containing Don Quaiprovided no relief.

Evaluation included a baseline morning urine sample and close assessmentof her symptoms of breast pain during the ensuing month. Following this,bioavailable DIM was begun as a supplement at 225 mg per day togetherwith increased dietary fiber. Complete resolution of the breast pain wasnoted following one month. A second urine sample was obtained after onemonth on the DIM supplement.

The urine samples were subsequently analyzed for their levels of2-hydroxy and 16-hydroxy estrogen metabolites, using an Elisa basedassay. (Estratest, Immunacare, Inc., Bethlehem, Pa.) These results ofbefore and after DIM supplementation breast pain scores and estrogenmetabolite testing are summarized below.

Indicator Monitored Before DIM After DIM Typical Breast Pain ScoreModerate Absent KEY: 0 = none 2/4 0/4 1 = mild; 2 = moderate; 3 =significant; 4 = severe Typical Breast Soreness Score Significant Absent3/4 0/4 Urinary Estrogen Metabolites (ng/ml/mg Creatinine): 2-Hydroxyestrone level 22.2  26.4  16-Hydroxy estrone level 7.3 5.1 2-Hydroxy to16-Hyroxy estrone  2.97  5.08 ratio Total urinary estrone metabolites28.9  31.3 

10. EXAMPLE Open Label Study of Oral Processed DIM in Women with BreastPain

20 women were referred by collaborating physicians for participation inan open label study of the use of a processed DIM dietarysupplementation for recurrent mastalgia. Individuals were selected whomet the criteria of recurrent bilateral breast pain for at least 6months. During the study, the participants avoided herbs and otherdietary supplements which might effect estrogen. This included avoidanceof Evening Primrose Oil, borage oil, soy isoflavones, Red Cloverextract, Don Quai, and Black Cohash. Using a breast pain calendar,participating individuals identified their level of pain, soreness, andswelling on separate analog pain scale scores for each category. 18 outof 20 participants showed some improvement in their symptoms which wasnoted from 10 days to 1 month after initiating supplementation. Of thoseparticipants showing improvement, 12 showed significant improvement withlevels dropping from moderate and severe to mild or absent. Theseimprovements in symptoms were seen at a dose of 150 to 300 mg/day of thesupplement which corresponds to a daily dose of 50-100 mg/day of DIM. Noparticipants reported side effects. Of further note was one participantaged 51 who had complete resolution of pain and noted the disappearanceof a breast cyst previously documented by her physician.

11. EXAMPLE Transdermal use of DIM for Treatment of Chronic Breast Pain

E. B. is a 38 year old woman with the recurrent, symptoms of bilateralpainful breasts. She describes the pain as bilateral, worse beforemenstrual periods and occurring every month beginning about 1 weekfollowing cessation of her menses. The pain is described as a sorenessassociated with movement and a feeling of heaviness or swelling of bothbreasts.

After maintaining a diary documenting breast pain for one menstrualcycle, E. B. began to apply a cosmetic formulation containing DIM. Afteronly one week of application of 5 cc of the cream nightly, completeresolution of the pain was noted. Also noted was a clear resolution inthe sense of swelling of the breasts. The cream was used consistentlyfor 2 months and resolution of the pain was documented on a breast paindiary. No side effects were experienced.

Following these two months, use of the cream was discontinued. Withinone month, the breast pain recurred although to a lesser severity thanwas documented before treatment.

12. EXAMPLE The Use of DIM for Endometriosis

P. M. is a slender and athletic woman of 32 years who soughtalternatives in her management of severe endometriosis. Her symptoms ofrecurrent mid-cycle and menstrually associated pain have been diagnosedas due to endometriosis based on pelvic laparoscopy. This procedureconfirmed aggressive endometriosis with ectopic endometrial implantsremoved from the pelvis and associated with intestinal serosal spread. Ahistory of 2 years f intense pelvic pain at mid-cycle and duringmenstrual flow was reported prior to the laparoscopy. Her mother gives ahistory of painful menstrual periods starting in her 20's. She went onto develop ovarian cancer requiring radical surgery at 56 years. Amaternal aunt developed cervical cancer requiring surgery. Both motherand aunt had a peri-menopausal history of chronic, recurrent breast painwith fibrocystic changes. Like the patient, a younger sister has beentroubled with painful menstrual periods and pelvic pain leading tolaparoscopy and the diagnosis of endometriosis.

Following the patient's laparoscopy, one menstrual period was stillassociated with significant pain. Supplementation with bioavailableprocessed DIM was begun approximately 6 weeks following the laparoscopy.Initial supplementation with bioavailable processed DIM provided 300mg/day for one month which was reduced to 150 mg/day thereafter. Sincestarting treatment, there was disappearance of pain at midcycle andimprovement of pain associated with menses. The patient continued DIMsupplementation for about one year. During this time regular menstrualperiods became more comfortable, no longer requiring analgesics. No sideeffects were reported.

Diagnosis of the endometriosis and reduction in pain severity correlatedwith serial levels of serum Ca-125 antigen. Ca-125 is a serum markerwith documented usefulness in monitoring the activity of endometriosisin a given patient (Pittaway, D. E., and Fayez, J. A., “The use ofCA-125 in the diagnosis and management of of endometriosis”, FertilSteril, 46, page 790, 1986) The following chart shows the association ofthis marker, useful as a measure of changes in the activity ofendometriosis.

Follow- Follow- Follow Patient Pre Post up Visit up Visit up VisitStatus Surgery Surgery #1 #2 #3 Use of DIM NO NO YES YES YES Ca-125 69.654.1 26.4 23.2 34.0 Antigen Pain Level Severe Moderate Improved ImprovedImproved

13. EXAMPLE Use of Transdermal and Processed DIM for the Therapy ofMastalgia with Associated Improvement in Cervical Dysplasia

V. H. is a 45 year old woman with a long history of fibrocystic breasts,recurrent severe breast pain, and cervical dysplasia. The breast painoccurs on a monthly basis during the second half of the menstrual cycleand requires the use of analgesics like ibuprofen. The breast paindiminishes with onset of the menses. Abnormal pap smears of the uterinecervix were first noted in her mid thirties. The cervical dysplasia wascategorized as Class I cervical intraepithelial neoplasia on a cervicalbiopsy taken approximately 1 year ago. V. H. began testing transdermalDIM in a 1.5% strength breast cream for relief of monthly breast pain.Dramatic resolution occurred over a period of 2 weeks. During this time,a reduction and disappearance of chronic vaginal discharge which hadbeen present and attributed to the cervical dysplasia were also noted.Following two weeks of transdermal use of DIM, V. H. began daily use oforal processed DIM at a dose of 50 mg per day of DIM. After two monthsof oral therapy, follow up pelvic examination revealed a more normalappearing cervix. No side effects were noted with the use of either DIMpreparation.

1. A method of treating endometriosis in a subject having endometriosiscomprising administering to the subject an amount of an indole selectedfrom the group consisting of 3,3′-Diindolylmethane (DIM),2-(Indol-3-ylmethyl)-3,3′-Diindolylmethane (LTr-1) and Indole-3-carbinol(I3C) effective to reduce one or more symptoms associated withendometriosis, wherein the indole is formulated as a tablet, pill,capsule, suppository, cream, parenteral suspension or liposomal spray,or is suspended as microparticles in a starch carrier matrix.
 2. Themethod according to claim 1, wherein the symptom is selected from thegroup consisting of pain, dysplasia, and the level Ca-125 antigendetectable in serum.
 3. The method according to claim 1, wherein theindole is I3C.
 4. The method of claim 1, wherein the indole is DIM. 5.The method of claim 4, wherein the DIM is suspended as microparticles ina starch carrier matrix.
 6. The method of claim 1, wherein the indole isLTR-1.
 7. The method of claim 1, further comprising administeringgrapefruit, grapefruit concentrate, grapefruit juice, or grapefruitjuice concentrate.
 8. The method of anyone of claim 3, 4 or 6, whereinthe indole is suspended as microparticles in a starch carrier matrix. 9.The method of claim 1, wherein the indole is formulated as a tablet. 10.The method of claim 1, wherein the indole is formulated as a pill. 11.The method of claim 1, wherein the indole is formulated as a capsule.12. The method of claim 1, wherein the indole is formulated as asuppository.
 13. The method of claim 1, wherein the indole is formulatedas a cream.
 14. The method of claim 1, wherein the indole is formulatedas a parenteral suspension.
 15. The method of claim 1, wherein theindole is formulated as a liposomal spray.
 16. The method of claim 1,wherein the indole is suspended as microparticles in a starch carriermatrix.
 17. The method of anyone of claims 9, 10 or 11, wherein theindole is administered orally.
 18. The method of anyone of claims 12, 13or 15, wherein the indole is administered by direct application to thevaginal or cervical mucosa of the subject.
 19. The method anyone ofclaims 12, 13 or 15, wherein the indole is administered transdermally.